Two small groups of people are of particular interest to researchers due to
their ability to remain disease free after exposure to HIV. The first group,
long-term nonprogressors, are clearly infected with virus but control virus
replication at low levels and have not progressed to disease. Within this
group, some individuals have barely detectable virus and are referred to as
elite controllers. The second group, highly exposed seronegative
individuals, have been repeatedly exposed to HIV yet remain disease free
and have no detectable virus. Intriguingly, some members of this latter
+
group appear to possess HIV-specific CD8 T-cells suggesting previous
exposure to the virus or at least to noninfectious viral antigens. Whether the
immune response seen in these individuals is responsible for clearing an
HIV infection is unclear. Nonetheless, these individuals are the focus of
much interest for vaccine design and development. We will now review key
aspects of the virus itself including its cellular tropism, genome and life
cycle.
HIV-1 genome
HIV-1 is a retrovirus, which means that it has an RNA genome but that
replication passes through DNA with the involvement of the enzyme
reverse transcriptase. It belongs to a group of retroviruses called the
lentiviruses, from the Latin lentus meaning “slow”, because of the slow
course of disease associated with infection by these viruses. The HIV-1
genome is composed of approximately 9 kb of RNA, which consists of nine
different genes encoding 15 proteins. Two copies of the single-stranded
genome are packaged in the virus particle along with additional enzymes
and accessory proteins. Three of the reading frames encode Gag (group
specific antigen), Pol (polymerase) and Env (envelope) polyproteins, which
are proteolytically cleaved into individual structural proteins and enzymes
(Figure 14.13). Gag is cleaved into four structural proteins, MA (matrix),
CA (capsid), NC (nucleocapsid) and p6, while Env is cleaved into two, SU
(surface gp120) and TM (transmembrane gp41). Pol cleavage produces the
enzymes PR (protease), RT (reverse transcriptase) and IN (integrase), which
are encapsulated in the virus particle. Several accessory proteins are also
encoded, three of which—Vif, Vpr and Nef—are packaged inside the virus
particle. The remaining accessory proteins are Tat, Rev and Vpu. The
functions of the 15 HIV proteins are summarized in Figure 14.13 and
discussed in relation to the HIV life cycle below.
Figure 14.13. The HIV-1 genome.
The organization of the genome is shown and the functions of the gene
products summarized. (Kindly provided by Warner Greene; after Greene
W.C. & Peterlin B.M. (2002) Nature Medicine 8, 673–680.)
The life cycle of HIV-1
Viral entry
Initial virus–cell attachment is believed to be mediated primarily through
nonspecific interactions between the envelope spikes that decorate the
surface of the virus and target T-cell surface molecules. The envelope spike
is a trimer of heterodimers composed of noncovalently associated surface
glycoprotein (gp120) and transmembrane glycoprotein (gp41) subunits. The
sugar moieties and positively charged patches on gp120 probably mediate
binding to cell surface lectins and negatively charged heparan sulfate
proteoglycans, respectively.
The first receptor-specific binding event occurs when gp120 on the viral
envelope spike engages CD4 on the target T-cell surface (Figure 14.14).
HIV-1 specifically infects cells expressing CD4, including T-lymphocytes,
macrophages and dendritic cells. CD4 binds with high affinity to a recessed
cavity of gp120 as revealed by a structure of gp120 in complex with CD4.
This binding event triggers multiple conformational changes in gp120 that
expose and form the coreceptor binding site. The coreceptor is most often
the chemokine receptor CCR5 or CXCR4. These receptors normally
function in chemoattraction, in which immune cells move along gradients
of chemokine molecules to sites of inflammation. HIV-1s are often grouped
by their coreceptor usage. R5 viruses use CCR5, X4 viruses use CXCR4
and dual tropic R5X4 viruses use both CCR5 and CXCR4. R5 viruses only
require low levels of CD4 expressed on the surface of target T-cells,
whereas X4 viruses require higher levels. Thus, differential expression of
CD4 and coreceptors makes different T-cell types (or subtypes) more
susceptible to infection by either X4 or R5 viruses: X4 viruses infect naive
+
CD4 T-cells and mature DCs while the preferred in vivo targets of R5
viruses include immature dendritic cells, macrophages and activated
+
effector or memory CD4 T-cells. Initially, R5 variants were labeled as
“macrophagetropic” when variants were classified based on the cell lines in
which they could grow in vitro and likewise, X4 viruses were labeled as
“lymphocyte-tropic.” These former designations for HIV variants are
misleading, as R5 viruses do infect lymphocytes, and therefore the
designations were changed to reflect coreceptor usage.
Coreceptor binding induces conformational changes in the
transmembrane glycoprotein, gp41, that result in the exposure of the highly
hydrophobic N-terminal fusion peptide of gp4l, previously buried in the
spike structure. The fusion peptide inserts into the host T-cell membrane
like a harpoon, both destabilizing the target T-cell membrane and generating
an extended α-helical gp4l fusion intermediate, designated the “pre-hairpin
intermediate.” This intermediate is unstable and readily collapses back onto
itself forming a six-helix bundle, or “hairpin,” comprising three internal a-
helices arranged antiparallel to three external a-helices. The only high-
resolution structure of gp4l available to date is of gp4l in this putative
postfusion form. The collapse of gp4l into this extremely stable six-helical
bundle is thought to provide the thermodynamic driving force for fusion.
Six-helical bundles are a common structural motif among other viral and
cellular fusion proteins; other viruses having surface proteins with structural
similarities to gp4l include influenza virus, SARS and Ebola virus.
Although it is not well understood how six-helical bundle formation enables
the merging of cellular and viral membranes, if bundle formation is
prevented using peptide analogs that compete for the occupancy of the
external a-helices, fusion is also abrogated. One such peptide has been
developed into an HIV drug, the first of a class of drugs referred to as viral
entry inhibitors.
Figure 14.14. Steps in the HIV replication cycle (courtesy of NIAID).
Fusion is a highly cooperative process that occurs on a time scale of
minutes and has been proposed to require the interaction of one to several
spikes with corresponding receptors and coreceptors to be an efficient
process.
Following fusion, the virus particle has lost its enveloped exterior, and the
viral core, or reverse transcription complex, remains. This complex is
composed of two viral RNAs, RT, IN, tRNA Lys , matrix (p17), nucleocapsid
(p7), capsid protein (p24) and Vpr.
Reverse transcription and integration
En route to the nucleus, RT uses the two single-stranded RNA molecules
enclosed within the viral core as a template to convert the viral genome into
a double-stranded cDNA copy of the viral genome. RT has no proofreading
mechanism and introduces approximately one mutation per genome per
reverse transcription. RNase H degrades the RNA template as the minus
strand DNA is synthesized and DNA polymerase catalyzes the generation
of a double-stranded viral cDNA genome.
Upon reverse transcription, the complex contains essentially the same
factors as before, except that the RNA genome has been replaced with a
newly synthesized cDNA genome. This complex is referred to as the
preintegration complex, which translocates to the nucleus, possibly via actin
filaments and microtubules by a mechanism only partially understood,
given the large size of the complex.
Integration of the viral cDNA genome into the host T-cell genome is
mediated by integrase and the actions of several host proteins (Figure
14.14). It requires the viral LTR sequence and is preferentially targeted to
areas of active transcription. Integration can lead to latent or
transcriptionally active viral cDNA referred to as a provirus. Active
provirus serves as the template for viral replication and transcription.
Latency explains the inability of viral therapies employed to date to
eliminate virus completely from infected individuals and is the great
challenge to a complete cure for HIV. The number of latently infected cells
6
5
in an infected individual is very small, of the order of 10 –10 .
Replication
Replication of the virus commences postintegration with the production of
nascent viral transcripts by cellular RNA polymerases (Figure 14.14).
Transcription is regulated by proteins that bind within the LTR sequences,
which flank the genome of the virus. For example, activation of T-cells
results in the expression of transcription factor NFκB. NFκB binds to
several promoters including those within the 5′-LTR.
Production of the viral proteins is biphasic. During the early phase (also
called the Rev-independent phase), the viral transcripts are completely
processed (i.e. all internal splice sites are utilized), polyadenylated and
exported to the cytoplasm as all other cellular transcripts. Translation of
these transcripts results in three gene products: Tat, Rev and Nef. Like other
viruses, HIV-1 makes full use of a single template and therefore, in order
for the other genes to be expressed, alternative splicing patterns are utilized
(four different 5′-splice sites, eight different 3′-splice sites); however, this
cannot occur until a critical threshold of Rev is achieved in the nucleus. A
nuclear localization signal in the N-terminus of Rev guides it back to the
nucleus post-translation with the help of cellular factor importin p. This
arginine-rich domain also serves as a binding site for an RNA target, the
Rev response element (RRE), which is located within the env intron of all
incompletely spliced mRNAs. Splicing of HIV transcripts by cellular
splicing factors is an inefficient process, and this allows time for Rev to
bind the RRE. Rev cooperatively multimerizes (up to 12 additional Rev
monomers) along the RNA and this Rev–RRE complex associates with
exportin/Crm-1 via a nuclear export signal in the C--erminus of Rev. This
allows for efficient transport of the partially spliced or unspliced transcripts
from the nucleus to the cytoplasm before the splicing factors are able to
process the transcripts.
These actions by Rev permit the second phase of gene expression to
commence and the partially spliced and unspliced mRNAs are translated
into Env, Vif, Vpr and Vpu and Gag and Gag–Pol, respectively. This is a
crucial adaptation on the part of the virus as transcripts with introns are
normally retained and degraded if they cannot be processed. Without Rev,
HIV-1 is not able to transport its genetic material (containing multiple
introns) to the cytoplasm where newly synthesized virus particles assemble;
indeed, in experiments in which Rev is removed from the genome, the
resulting virus clones are replication incompetent.
Tat and Nef are also crucial in HIV replication. In the absence of Tat,
transcription begins but the polymerase fails to elongate efficiently along
the viral genome. Tat binds to a well defined structure on the RNA, recruits
positive elongation factors and promotes the rate of viral replication. Nef
acts differently to Tat and Rev; it does not bind directly to viral RNA but
rather acts upon the environment of the infected cell to favor replication.
The activities of Nef include the ability to affect signaling cascades,
downregulate CD4 expression at the infected cell surface and promote the
generation of more infectious virions as well as virus dissemination. In
addition by downregulating MHC class I molecules from the cell surface,
Nef impairs immunological responses to HIV and inhibits apoptosis,
thereby prolonging the life of the infected cell and increasing viral
replication.
The number of mechanisms by which HIV promotes its own reproduction
is staggering. It reflects the rapid turnover and inherent infidelity in HIV
replication. The virus has sampled a huge number of different protein–
protein and protein–nucleic acid interactions in its dance with humans and
selection pressure has brought forth those interactions that favor virus
survival and expansion. This is evolution on a time scale far shorter than
normally experienced.
Virus assembly, budding and maturation
New virus particle assembly occurs at the plasma membrane of the infected
cell (Figure 14.4). One of the viral proteins translated in the cytosol during
the late phase of gene expression is the Gag precursor protein p55. p55
traffics to the plasma membrane or late endosomes and attaches to lipid
bilayer where Env glycoproteins are attached via the transmembrane anchor
of gp41. Assembly is dependent on the cellular protein HP68 that binds p55
and promotes the formation of an immature viral core. Other structural viral
proteins assemble at the cell membrane with two copies of the viral RNA
genome, RT, protease and integrase to be packaged into an immature virus
particle. One of the key structural proteins present is p6, which connects the
virus core to components of the endosomal sorting complex at sites of
budding in the plasma membrane and late endosomes. Just before budding,
other host factors including cytoplasmic viral restriction factors such as
APOBEC3G can be incorporated into the virion. Coincident with budding
of the immature virion from the plasma membrane, proteolytic processing
of capsid occurs, generating the mature viral particle.
APOBEC3G is an interesting molecule that can restrict viral replication
by cysteine deamination of DNA and resultant loss of functionality of viral
genomes. The HIV-1 protein vif binds to APOBEC3G and by targeting it
for professional degradation reduces its incorporation into virions.
APOBEC3G is expressed in primary cells such as lymphocytes and
macrophages and, as a consequence, Vif is essential for viral replication in
these cells.
Another important HIV-1 restriction factor is TRIM5α, which is
responsible for the resistance of primate cells to diverse retrovirus infection.
It targets the capsid protein and blocks an early step of retroviral infection
prior to reverse transcription. Finally, tetherin is a molecule that can
suppress virus release from infected cells—its action can be counteracted
by the HIV-1 protein Vpu.
In closing, it is important to note that much propagation of infection in
HIV-1 in vivo probably occurs by cell-to-cell spread of virus rather than by
free virus particles. Env proteins on the infected cell surface engage
receptors on neighboring target T-cells, but HIV-1 transfer still requires
viral budding. It appears that HIV-1 particles transfer directionally through
sites of contact between infected and uninfected T-cells in an arrangement
that has been termed the virological synapse with similarities to the
immunological synapse found between T-cells and DCs. Nef promotes the
formation of such synapses between infected macrophages and T-cells.
Vaginal transmission of HIV and the early stages
of infection
Most HIV infections are now acquired through heterosexual transmission,
most frequently by women through vaginal intercourse. There has therefore
been an increased focus on understanding how vaginal transmission takes
place and how one might intervene to prevent transmission. The SIV/
monkey model has been very useful in this area (Figure 14.15). It appears
that the virus struggles against the odds in the early phases of infection, but
once it gains a foothold, circumstances rapidly change in favor of the virus
to the point that progression to disease is virtually inevitable without drug
intervention.
The first problem the virus encounters is the mucosal barrier. If this
barrier is damaged, e.g. by ulcerative genital diseases, bacterial vaginosis or
after the use of some microbicides such as nonoxynol-9, then transmission
rates are increased. If the barrier is largely intact, then very few viruses will
make it across, probably via small breaks or by transport on DCs. DCs
express C-type lectins, such as DC-SIGN and DC-SIGNR, which bind high
mannose glycans displayed on the surface of gp120, thereby capturing
virions that may be internalized into a low-pH, nonlysosomal compartment,
where they remain infectious. Once across the barrier, free virus infects
+
target T-cells such as CD4 T-cells, macrophages and DCs in the lamina
+
propria. Infectious virus inside DCs can enter resting and activated CD4 T-
cells via DC–T-cell conjugates as bursts of viral replication are observed at
DC–T-cell synapses. In addition, HIV-1 Nef-induced upregulation of DC-
SIGN and β-chemokines in DCs may promote lymphocyte clustering and
viral spread. Other studies suggest that Nef may also alter the physiological
characteristics of infected macrophages so as to enhance conditions for viral
dissemination.
Nevertheless, at this point in time, there is only a small founder
population of infected cells, which must spread infection to the relatively
few in number and spatially dispersed susceptible cells in the mucosa.
Infection is still fragile and probably susceptible to intervention at this time.
Then sometime between a day and a week, the virus finds its way to
+
lymphoid tissue, a rich source of activated CD4 T-cells. Now conditions
favor the virus as access is provided to large numbers of closely packed
target T-cells leading to an extremely rapid rise in virus production to give
peak viral loads in plasma. One very important lymphoid tissue
compartment is the lamina propria of the gut where massive killing of
+
+
CD4 memory T cells occurs either via direct killing or via apoptosis. The
counter-attack by the host’s immune system has been described as “too
little, too late.”
HIV-1 therapy
Great advances have been made in recent years in the containment of HIV
replication in infected individuals and the slowing down or blocking of the
progression to AIDS. Many new drugs are available. Many steps in the
virus life cycle are potential targets for drugs, including: (i) entry; (ii)
fusion; (iii) reverse transcription; (iv) integration; (v) transcription/
transactivation; (vi) assembly; and (vii) maturation.
Currently, five classes of drugs targeting four steps are in clinical use. The
first antiretroviral class to become available was the nucleoside/nucleotide
reverse transcription inhibitors. These nucleoside/nucleotide analogs are
incorporated into the growing strand of viral DNA leading to chain
termination and the production of noninfectious virus. Reverse transcription
can also be inhibited by a second class of drugs, the non-
nucleoside/nucleotide reverse transcription inhibitors, which bind
allosterically to a site distant from the substrate-binding site. Viral protease
inhibitors inhibit cleavage of the gag and pol polyproteins. The first fusion
inhibitor, enfuvirtide, was approved by the Federal Food and Drug
Administration (FDA) in the USA in 2003, and is a peptide that binds to
gp41 to inhibit fusion. The first integrase inhibitor was approved in the
USA in 2007.
A major problem in HIV therapy is the development of drug resistance.
The error-prone nature of reverse transcription, the large viral load and the
rapid rate of virus replication in many infected individuals means that they
typically harbor a very large number of HIV variants. Administration of
drugs may select for a variant that has resistance. Drug resistance against
many protease inhibitors and some of the more potent nucleoside analogs
can develop within a few days as a single mutation in the target enzyme
confers resistance to many of these drugs. Resistance to other
antiretrovirals, such as zidovudine (AZT), requires multiple mutations
(three or four for AZT) and correspondingly longer to develop. Due to the
relatively rapid development of resistance to all HIV drugs used singly,
successful suppression of HIV currently necessitates combination therapy.
Antiretroviral therapy (ART) typically involves the administration of a
combination of drugs operating by different mechanisms.
Figure 14.15. Time frame, sites and major events in vaginal
transmission of HIV/SIV.
The SIV rhesus macaque animal model provides a window through which
to view early infection. Within hours, virus in the inoculum may gain access
through breaks in the mucosal epithelial barrier to susceptible target cells.
The small focal infected founder population is initially composed mainly of
infected resting CD4 T-cells. The founder population expands locally in
these “resting” and in activated CD4 T-cells. Local expansion is necessary
to disseminate infection to the draining lymph node and subsequently
through the bloodstream to establish a self-propagating infection in
secondary lymphoid organs. Crossing the barrier, small founder populations
(with the associated risk that the basic reproductive rate, R , will fall below
0
one), and local expansion are vulnerabilities for the virus in week 1 of
infection. These vulnerabilities create opportunities for prevention of
infection. Microbicides and vaccines that could reduce the size of the
founder populations of infected cells might abort infection at the point of
entry or prevent the efficient viral seeding of distant sites required for the
establishment of a systemic infection. In humans, HIV-1 infection is first
clinically manifest in the time frame of weeks and hence the need for the
animal model to view sexual mucosal transmission and the earlier stages of
infection. (Kindly provided by Ashley Haase; after Haase A.T. (2010)
Nature 464, 217–223.)
ART has proven very effective in the management of viral levels in
infected individuals. During the first 2 weeks of treatment, plasma virus
loads decrease very rapidly reflecting the inhibition of virus production
from infected cells and the rapid clearance of free virus from the circulation
(half-life about 30 min). The results indicate that the half-1ife of
productively infected cells is about 2 days. At the end of 2 weeks, viral
plasma levels have decreased by more than 95%, signifying a nearly
+
complete loss of productively infected CD4 T-cells. There is a
+
concomitant rise in CD4 T-cell counts in the peripheral blood as HIV
replication and infection is controlled. This rise has been attributed to three
+
mechanisms: redistribution of CD4 memory cells from lymphoid tissues
into the circulation; reduction in the abnormal levels of immune activation
+
associated with reduced CD8 T-cell killing of infected cells; and the
emergence of new naive T-cells from the thymus.
Figure 14.16. Model for neutralization of HIV by antibody.
Viral entry is mediated by the interaction of envelope spikes on the virus
surface with CD4 and CCR5 on the target cell surface. The antibody
molecule (Ab) has a molecular volume approaching that of a spike.
Therefore the attachment of an antibody molecule to a spike is expected to
show strong steric interference with virus attachment and/or fusion. (After
Poignard et al. (2001) Annual Review of Immunology, 19, 253–274; and
Schief et al. (2010) Current Opinion in HIV and AIDS 4, 431–440).)
After the initial rapid and almost complete clearance of free virus, a
second slow phase of viral decay reflects the very slow decay of virus
production in longer-lived reservoirs, such as in DCs and macrophages,
+
from latently infected memory CD4 T-cells that have been activated. A
third phase has been postulated, which is even slower, resulting from
reactivation of integrated provirus in memory T-cells and other long-lived
reservoirs of infection. Follicular DCs store virus in the form of immune
complexes, making them potential long-term sources of infectious virus.
These latent reservoirs may persist for years and are resistant to current
HIV drug therapy.
HIV-1 vaccines
Most epidemiologists agree that the most efficient means to control the
HIV-1 pandemic would be an effective vaccine. Unfortunately, the
development of such a vaccine faces some major hurdles intimately
associated with features of the virus. These include the variability of the
virus, the nature of the envelope spikes of the virus and the ability of the
virus to integrate into host chromosomes and become latent.
Most viral vaccines appear to be effective because they mimic natural
infection and elicit neutralizing antibody responses. Long-lived plasma cells
in the bone marrow secrete neutralizing antibodies that are present in serum
and can act immediately to inactivate virus particles (Figure 14.16). Indeed,
the likelihood that a vaccine will be effective is often assessed by looking at
serum neutralizing antibody levels. Additionally on contact with virus,
vaccine-induced memory B-cells are stimulated to secrete neutralizing
antibodies. Studies in monkeys show that neutralizing antibodies can
protect against HIV. If neutralizing antibodies are administered systemically
and then the monkeys challenged with a hybrid human (HIV)/ monkey
(SIV) virus, they show no signs of infection, i.e. they exhibit sterilizing
immunity. However, there is a requirement that the neutralizing antibodies
elicited by vaccination be active against a wide spectrum of different HIV
variants (so-called broadly neutralizing antibodies). Such antibodies are
known to exist but the design of immunogens to elicit them has not yet been
achieved. Indeed, natural HIV infection elicits relatively weak broadly
neutralizing antibody responses, highlighting the difficulties of finding an
appropriate immunogen. Natural infection tends to elicit type-specific
neutralizing antibodies (Figure 14.17). When these antibodies reach a
critical threshold, a resistant virus emerges. Eventually, a neutralizing
antibody response to this virus develops and a new resistant virus emerges
and so on. Apparently the virus always stays one step ahead of the
neutralizing antibody response.
For the reasons outlined above, it appears that it will be challenging to
design an HIV vaccine that will provide sterilizing immunity through
elicitation of broadly neutralizing antibodies. In fact, most current vaccines
effective against other viruses are not thought to provide sterilizing
immunity. Rather they elicit sufficient serum titers of neutralizing antibody
to blunt infection, which is then contained by cellular or innate immunity
and overt symptoms are avoided. In other words, vaccination protects
against disease rather than infection.
Figure 14.17. Evolution of the neutralizing antibody response in HIV
infection.
A–E refer to virus and sera from time points A–E during the course of
infection of an individual. Serum taken at time point A has no significant
neutralizing activity against virus isolated from the plasma of the infected
individual at time point A. Serum taken at time point B has some weak
activity. Serum taken at time point C and points thereafter clearly
neutralizes virus from time point A. Once the serum neutralizing antibody
concentration has reached a certain threshold following exposure to a given
predominant virus variant, selection pressure is exerted such that a new
neutralization-resistant variant emerges from the huge pool of variants
present in the infected individual. A neutralizing antibody response
develops to this new variant and the cycle is repeated. (Courtesy of Doug
Richman; after Richman D.D. et al. (2003) Proceedings of the National
Academy of Sciences of the USA 100, 4144–4149.)
Studies in animal models have shown that protection against disease for a
number of viruses can be achieved by eliciting a cellular immune response
through vaccination. In the absence of effective methods to elicit broadly
neutralizing antibodies, much HIV vaccine research has targeted cellular
immune responses. The primary rationale has been that if potent T-cellular
immune responses can be elicited in vaccinees, the response may reduce the
+
damage to CD4 T-cells following primary infection and lower the viral set
point. As viral set point has been correlated with time of progression to
AIDS, this would provide direct benefit to vaccinees. Furthermore,
reduction of average plasma viral loads in vaccinated individuals should
reduce transmission rates since transmission correlates with plasma viral
load. Thus, vaccination should provide benefit to the population at large.
+
Finally, reducing the damage to CD4 T-cells in primary infection may help
to maintain immunity against many pathogens over a long period.
Most studies on so-called “T-cell vaccines” have been carried out in
+
monkeys. The results have been mixed. The best CD8 T-cell responses, at
least in terms of ELISPOT measurements, have been achieved using
recombinant viral vectors to express HIV/SIV gene products. In particular,
adenovirus vectors, either alone or in combination with other vectors or
DNA vaccination, have elicited significant T-cellular responses. These
responses have shown some protection in some monkey models but not in
others.
Four larger-scale human HIV vaccine trials have been carried out. Two
trials reporting in 2003 were based on recombinant monomeric gp120 and
could be described as “antibody vaccines” in that they were expected to
elicit primarily antibody responses. However the responses did not
neutralize typical HIV isolates and the vaccines showed no efficacy. A trial
reporting in 2007 was based on an adenovirus vector encoding HIV internal
proteins gag, pol and nef and was described as a “ T-cell vaccine.” The
vaccine showed no efficacy. Initially, it was thought that the vaccine had
enhanced infection rates but detailed studies have brought this interpretation
into question. The most recent trial reporting in 2009 was based on a
canarypox vector encoding HIV gag, pro and env with boosting by env
(recombinant gp120). This has been described as an “antibody and T-cell
vaccine.” The trial described possible modest efficacy close to the limits of
statistical significance that appeared to have very short duration. Intensive
efforts are ongoing to see if any correlate of protection in this trial can be
identified.
Overall, it is clear that the development of an HIV vaccine is one of the
major challenges facing modern medicine. Many believe that success will
require the development of immunogens that can elicit both potent broadly
neutralizing antibody and cellular immune responses.
SUMMARY
Primary immunodeficiency diseases (PIDs)
Primary immunodeficiencies are much less common than secondary,
occur as a result of a gene defect, and can affect almost any component
of the immune response.
They are characterized by opportunistic infections.
Several X-linked mutations produce PIDs in males.
PIDs illuminate the importance of individual components of the
immune system in combating particular infectious agents.
Treatment includes prophylactic antibiotics, intravenous Ig, bone
marrow transplantation and, potentially, gene therapy.
PIDs affecting innate responses
Mutations in the genes encoding pattern recognition receptors or
their associated adaptor and signaling molecules will particularly
affect innate responses.
Phagocytic cell or complement defects result in infection with
bacteria that would normally be disposed of by opsonization and
phagocytosis.
Where there is an inability to produce the membrane attack complex
there is only a very limited spectrum of increased infections, mainly
with Neisseria spp.
Defects in complement components are associated with age-related
macular degeneration or systemic lupus erythematosus.
A mutation in any one of several genes involved in the IFNγ
response leads to increased susceptibility to mycobacterial infections.
Mutations that influence TNF pathways can lead to conditions in
which inflammation occurs in the absence of a stimulus.
B-cell primary immunodeficiencies
Selective IgA deficiency is the most common PID but affected
individuals are often symptomless.
In X-linked agammaglobulinemia all classes of antibodies are absent
or only present at extremely low concentrations due to a defect in the
Bruton’s tyrosine kinase resulting in maturation arrest at the pre-B-cell
stage.
Common variable immunodeficiency is associated with low IgG and
IgA and/or IgM.
T-cell primary immunodeficiencies
Patients with T-cell deficiencies are susceptible to intracellular
bacteria, viruses and fungi.
A lack of functional T-cells will impair B-cell responses.
In complete DiGeorge syndrome the absence of a thymus leads to an
inability to produce T-cells, although in most cases there is only a
partial defect.
Mutations affecting the enzyme purine nucleoside phosphorylase
lead to the accumulation of toxic metabolites that particularly affect T-
cells.
The genes linked to Omenn syndrome are similar to those
responsible for SCID but the site of the actual mutation is different and
does not have quite such a profound effect.
An absence of either MHC class I or class II molecules will result in
the inability of T-cells to undergo positive selection in the thymus.
A number of gene defects, including those associated with Wiskott–
Aldrich syndrome and with hyper-IgM syndrome, adversely affect the
ability of T-cells to interact with B-lymphocytes.
Mutations in genes required for regulatory T-cell activity result in
autoimmune conditions.
Severe combined immunodeficiency
Null mutations in a number of different genes, including γC, ADA,
RAG-1, RAG-2, JAK-3, Artemis and the IL-7R α chain, can result in
SCID.
There is a complete block in the development of T-cells, and thus
complete lack of help for B-cells. Depending on the particular gene
defect, B-cells and/or NK cells may also be absent.
Most cases of gene therapy for PIDs have attempted to insert a
normal gene for ADA or γC.
Secondary immunodeficiency
Immunodeficiency may arise as a secondary consequence of
malnutrition, lymphoproliferative disorders, agents such as X-rays and
cytotoxic drugs, and viral infections.
Acquired immunodeficiency syndrome (AIDS)
AIDS results from infection with the lentiviruses HIV-1 or HIV-2,
with HIV-1 being much more prevalent worldwide.
+
+
HIV-1 infects CD4 cells, including CD4 T-cells, macrophages
and dendritic cells.
+
Depletion of CD4 T-cells, dramatically in primary infection
particularly in the gut and then more slowly over a period of years
during clinical latency, leads to damage to the immune system, which
renders an individual susceptible to opportunistic pathogens (AIDS).
HIV-1 is a retrovirus, which gains entry to cells by interaction of
envelope spikes with CD4 and the chemokine receptors, CCR5 or
CXCR4. The RNA genome is reverse transcribed and the resulting
viral cDNA integrated into host T-cell chromosomes.
Integrated proviral DNA can remain latent in cells for very long
times, posing enormous problems for complete elimination of the virus
from an individual and therefore hampering a complete cure for HIV-1
infection.
Proviral DNA can be transcribed to generate new viral particles with
the aid of several viral accessory proteins, which act to aid viral
replication and/or adapt the host T-cell machinery to virus production.
A major hallmark of HIV is the enormous diversity of the virus,
present even in a single infected individual, because of the inherent
errors involved in transcribing from an RNA genome, the rapid
turnover of the virus and the high viral burden typically carried by the
individual.
Viral diversity and latency present major challenges to drug therapy
but nevertheless drug design has been highly successful and
combination drug regimes can hold the virus in check for many years,
if not indefinitely.
Vaccine design has also struggled with viral diversity and no
immunogens that elicit broadly neutralizing antibodies or sufficiently
potent T-cell responses to significantly contain challenge with a wide
diversity of HIVs have yet been designed, although efforts are intense
and there are promising leads.
FURTHER READING
Arhel N. & Kirchhoff F. (2010) Host proteins involved in HIV infection:
new therapeutic targets. Biochimica et Biophysica Acta 1802, 313–321.
Austen K.F., Burakoff S.J., Rosen F.S. & Strom T.B. (eds) (2001)
Therapeutic Immunology, 2nd edn. Blackwell Science, Oxford.
Bonilla F.A. & Geha R.S. (2006) Update on primary immunodeficiency
diseases. Journal of Allergy and Clinical Immunology 117 (2 suppl), S435–
441.
Broder S. (2010) The development of antiretroviral therapy and its impact
on the HIV-1/AIDS pandemic. Antiviral Research 85, 1–18.
Buckley R.H. (2002) Primary immunodeficiency diseases: dissectors of the
immune system. Immunological Reviews 185, 206–219.
Conley M.E. et al. (2009) Primary B cell immunodeficiencies: comparisons
and contrasts. Annual Review of Immunology 27, 199–227.
Greene W.C. & Peterlin B.M. (2002) Charting HlV’s remarkable voyage
through the cell: basic science as a passport to future therapy. Nature
Medicine 8, 673–680.
Haase A.T. (2010) Targeting early infection to prevent HIV-1 mucosal
transmission. Nature 464, 217–223.
Klasse P.J., Shattock R. & Moore J.P. (2008) Antiretroviral drug-based
microbicides to prevent HIV-1 sexual transmission. AnnualReview of
Medicine 59, 455–471.
Kohn D.B. ( 2010 ) Update on gene therapy for immunodeficiencies.
Clinical Immunology 135, 247–254.
Malim M.H. & Emerman M. ( 2008 ) HIV-1 accessory proteins—ensuring
viral survival in a hostile environment. CellHost & Microbe 3, 388–398.
McMichael A.J., Borrow P., Tomaras G.D., Goonetilleke N. & Haynes B.F.
(2010) The immune response during acute HIV-1 infection: clues for
vaccine development. NatureReviewsImmunology 10, 11—23.
Notarangelo L.D. et al. (2009) Primary immunodeficiencies: 2009 update.
International Union of Immunological Societies Expert Committee on
Primary Immunodeficiencies, Journal of Allergy and Clinical Immunology
124, 1161—1178.
Ochs H.D., Smith C.I.E. & Puck J.M. (eds.) (2007) Primary
Immunodeficiency Diseases—A Molecular and Genetic Approach. 2nd edn.
Oxford University Press, Oxford.
Richman D.D., Margolis D.M., Delaney M., Greene W.C., Hazuda D. &
Pomerantz R.J. (2009) The challenge of finding a cure for HIV infection.
Science 323,1304–1307.
Sharp P.M. & Hahn B.H. (2008) AIDS: prehistory of HIV-1. Nature 455,
605–606.
Simonte S.J. & Cunningham-Rundles C. ( 2003 ) Update on primary
immunodeficiency: defects of lymphocytes. Clinical Immunology 109, 109–
118.
Tilton J.C. & Doms R.W. (2010) Entry inhibitors in the treatment of HIV-1
infection. Antiviral Research 85, 91–100.
Turvey S.E., Bonilla F.A. & Junker A.K. (2009) Primary immunodeficiency
diseases: a practical guide for clinicians. PostgraduateMedicalJournal 85,
660–666.
van de Vosse E., van Dissel J.T. & Ottenhoff T.H. (2009) Genetic
deficiencies of innate immune signalling in human infectious disease. The
Lancet Infectious Diseases 9, 688–698.
Virgin H.W. & Walker B.D. (2010) Immunology and the elusive AIDS
vaccine. Nature 464, 224–231.
Walker L.M. & Burton D.R. (2010) Rational antibody-based HIV-1 vaccine
design: current approaches and future directions. Current Opinion in
Immunology 22, 358–366.
Now visit www.roitt.com to test yourself on this chapter.
CHAPTER 15
Allergy and other hypersensitivities
Key Topics
Anaphylactic hypersensitivity (type I)
Antibody-dependent cytotoxic hypersensitivity (type II)
Immune complex-mediated hypersensitivity (type III)
Cell-mediated (delayed-type) hypersensitivity (type IV)
An addition to the original classification—stimulatory hypersensitivity
(“type V”)
Just to Recap ...
Infections are dealt with by appropriate immune responses that detect
foreign antigens. In the case of adaptive responses there is a necessity for
clonal proliferation of lymphocytes in order to generate sufficient numbers
of antigen-specific cells. Antibody of a class appropriate to clear the
infection is produced and binds to the surface of the pathogen. The
formation of IgM- or IgG- containing immune complexes triggers the
activation of the classical complement pathway. IgG and complement
components opsonise microorganisms for subsequent phagocytosis. In the
case of parasitic infections, Th2-derived IL-4 and IL-13 encourage IgE
production by B-cells. Intracellular pathogens are dealt with by NK cells,
cytotoxic T-cells and by Th1 cells producing macrophage activating factors
such as IFNγ.
Introduction
In allergy the immune response extends beyond its usual boundary of
recognizing only foreign pathogens to also encompass what should be
innocuous environmental antigens. This is a form of hypersensitivity,
overzealous immunity that can also take the form of reactivity to antigens
from the same or different species. Such responses lead to tissue damage,
immunopathology, if the antigen is present in relatively large amounts or if
the acquired immune response is at a heightened level. It should be
emphasized that the mechanisms underlying hypersensitivity reactions are
the same as those normally employed by the body in combating infection.
The various hypersensitivity states were originally classified into types I–IV
by Gell and Coombs and this classification remains broadly useful.
However, it is often the case that in a particular disease state more than one
of these types coexist.
Milestone 15.1—The Discovery of
Anaphylaxis
Hypersensitive reactions in some individuals to normally innocuous environmental agents
have been observed from time immemorial. Scientific interest in such reactions was aroused
by the observations of Charles Richet and Paul Portier. During a South Sea cruise on Prince
Albert of Monaco’s yacht, the Prince, presumably smarting from an encounter with Physalia
(the jellyfish known as the Portugese man-of-war with very nasty tentacles), suggested that
toxin production by the jellyfish might be of interest. Let Richet and Portier take up the
story in their own words (1902):
“On board the Prince ‘s yacht, experiments were carried out proving
that an aqueous glycerin extract of the filaments of Physalia is
extremely toxic to ducks and rabbits. On returning to France, I could not
obtain Physalia and decided to study comparatively the tentacles of
Actiniaria (sea anemone). While endeavouring to determine the toxic
dose (of extracts), we soon discovered that some days must elapse
before fixing it; for several dogs did not die until the fourth or fifth day
after administration or even later. We kept those that had been given
insufficient to kill, in order to carry out a second investigation upon
these when they had recovered. At this point an unforeseen event
occurred. The dogs that had recovered were intensely sensitive and died
a few minutes after the administration of small doses. The most typical
experiment, the one in which the result was indisputable, was carried
out on a particularly healthy dog. It was given at first 0.1 ml of the
glycerin extract without becoming ill: 22 days later, as it was in perfect
health, I gave it a second injection of the same amount. In a few seconds
it was extremely ill; breathing became distressful; it could scarcely drag
itself along, lay on its side, was seized with diarrhea, vomited blood and
died in 25 minutes.”
The development of sensitivity to relatively harmless substances was
termed by these authors anaphylaxis, in contrast to prophylaxis.
Anaphylactich ypersensitivity (type I)
The phenomenon of anaphylaxis
The earliest accounts of inappropriate responses to foreign antigens relate to
anaphylaxis (Milestone 15.1). The phenomenon can be readily reproduced
in guinea-pigs that, like humans, are a highly susceptible species. A single
injection of 1 mg of an antigen such as egg albumin into a guinea-pig has
no obvious eff ect. Repeat the injection 2 – 3 weeks later and the sensitized
animal reacts very dramatically with the symptoms of generalized
anaphylaxis; almost immediately, the guinea-pig begins to wheeze and
within a few minutes dies from asphyxia. Examination shows intense
constriction of the bronchioles and bronchi and generally there is: (i)
contraction of smooth muscle; and (ii) dilatation of capillaries. Similar
reactions occur in human subjects highly allergic to insect stings, pollens,
foods, drugs such as penicillin, or other agents that have the potential to
cause life-threatening anaphylactic responses. In many instances only a
timely injection of epinephrine, which rapidly reverses the action of
histamine on smooth muscle contraction and capillary dilatation, can
prevent death. Individuals known to be at risk are given self administration
preloaded epinephrine syringes.
Sir Henry Dale recognized that histamine mimics the systemic changes of
anaphylaxis and, furthermore, that exposure of the uterus from a sensitized
guinea-pig to antigen induces an immediate contraction associated with an
explosive degranulation of mast cells (see Figure 1.20) responsible for the
release of histamine and a number of other mediators (see Figure 1.21).
Anaphylaxis is triggered by clustering of IgE
receptors on mast cells through cross-linking
In rodents two main types of mast cell have been recognized, those in the
intestinal mucosa and those in the peritoneum and other connective tissue
sites. Th ey diff er in a number of respects, for example in the type of
protease and proteoglycan in their granules, and in their ability to
proliferate and diff erentiate in response to stimulation by interleukin 3 (IL-
3) (Table 15.1). Th e two types have common precursors and are
interconvertible depending upon the environmental conditions, with the
mucosal MC ( tryptase) phenotype favored by IL-3 and connective tissue
t
MC (both tryptase and chymase) being promoted by relatively high levels
tc
of stem cell factor (c-kit ligand). In humans most mast cells in the intestinal
mucosa and lung alveoli are tryptase-only positive, whereas those in skin,
intestinal submucosa and other connective tissues are tryptase, chymase and
carboxypeptidase positive. A third, less frequent, population is chymase-
only positive and is found in the nasal mucosa and intestinal submucosa.
Mast cells, and their circulating counterpart the basophil, abundantly
10
display the Fc ε RI high-affinity ( K 10 M−1) receptor for IgE (cf. Table
a
3.2). The receptor is also expressed, albeit at considerably lower levels, on
Langerhans ’ cells, dendritic cells, monocytes, macrophages, neutrophils,
eosinophils, platelets and the intestinal epithelium. On basophils and mast
cells the receptor is a tetramer consisting of an α chain, a tetraspan β chain
and two disulfi de-linked γ chains, whereas on other cell types, where the
receptor is involved in antigen presentation rather than triggering
degranulation, the β chain is absent and therefore the receptor is a trimer.
The α chain possesses two external lg-type domains responsible for binding
the Cε3 region of IgE (Figure 15.1), whereas the γ chains and β chain each
contain a cytoplasmic immunoreceptor tyrosine-based activation motif
(ITAM) for cell signaling. In the absence of bound IgE the level of Fc ε RI
drops substantially. However, in its presence there is upregulation of the
receptor on mast cells and, because the γ chain is shared with the mast cell
Fc γ RIIIA, a consequent competitive downregulation of the Fc receptor for
IgG. Anaphylaxis is mediated by the reaction of the allergen with the IgE
antibodies held on the surface of the mast cell, cross-linking of these
antibodies triggering mediator release (Figure 15.2). Th e critical event is
aggregation of the receptors by cross-linking as clearly shown by the ability
of divalent antibodies reacting directly with the receptor to trigger the mast
cell.
Table 15.1. Comparison of the two main types of mast cell.
Characteristics Mucosal mast cell Connective tissue mast cell
General
Abbreviation* MC t MC tc
Distribution Gut & lung Most tissues**
Differentiation favored by IL-3 Stem cell factor
T-cell dependence + –
4
5
High affinity Fce receptor 2 × 10 /cell 3 × 10 /cell
Granules
Alcian blue and Safranin staining Blue & brown Blue
Ultrastructure Scrolls Gratings/lattices
Protease Tryptase Tryptase & chymase
Proteoglycan Chondroitin sulfate Heparin
Degranulation
Histamine release + ++
LTC : PGD release 25 : 1 1 : 40
2
4
Blocked by disodium cromoglycate/theophylline – +
* Based on protease in granules.
** Predominate in normal skin and intestinal submucosa.
Aggregation of the FcεRI α chains activates the Lyn and Fyn protein
tyrosine kinases associated with the β chains and, if the aggregates persist,
this leads to transphosphorylation of the β and γ chains of other Fc ε RI
receptors within the cluster and recruitment of the Syk kinase (Figure 15.3).
Th e subsequent series of phosphorylation-induced activation steps
ultimately leads to mast cell degranulation with release of preformed
mediators and the synthesis of arachidonic acid metabolites formed by the
cyclo-oxygenase and lipoxygenase pathways (cf. Figure 1.21). To
recapitulate, the preformed mediators released from the granules include
histamine, heparin, tryptase, chymase, carboxypeptidase, eosinophil,
neutrophil and monocyte chemotactic factors, platelet activating factor and
4
4
4
serotonin. By contrast, leukotrienes LTB , LTC and LTD , the
2
prostaglandin PGD and thromboxanes are all newly synthesized. The Th2-
type cytokines IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, as well as IL-1, IL-3,
IL-8, IL-11, granulocyte– macrophage colony- stimulating factor (GM-
CSF), TNF (tumor necrosis factor), CCL2 ( monocyte chemotactic protein-
1, MCP-1), CCL5 (RANTES) and CCL11 (eotaxin), are all also released.
Under normal circumstances, these mediators help to orchestrate the
development of a defensive acute inflammatory reaction (and in this context
let us not forget that complement fragments C3a and C5a can also trigger
mast cells through complement receptors). When there is a massive release
of these mediators under abnormal conditions, as in atopic disease, their
bronchoconstrictive and vasodilatory eff ects predominate and become
distinctly threatening.
Atopic a llergy
The a llergy march
Food allergy, eczema (atopic dermatitis), hayfever (seasonal allergic
conjunctivitis and rhinitis) and asthma often occur in the same individual.
Indeed in many individuals allergies develop in an ordered sequence that
has been referred to as the “ allergy march ” (Figure 15.4). Thus
gastrointestinal and cutaneous allergies developing early in life can be
followed later on by asthma and hayfever.
Figure 15.1. The structural basis of the binding of IgE to the high-affi
nity mast cell receptor FcεRI.
Side view of the complex with the two Fc chains in yellow and red and the
FcεRI α chain in blue; carbohydrate residues are shown as sticks. The two
Cε3 domains of the heavy chain dimer of IgE bind asymmetrically to two
distinct interaction sites on the a chain of the receptor. The (β-turn loop on
one Cε3 binds along one side of the α2 domain, while surface loops plus the
Cε2–Cε3 linker region on the other Cε3 interact with the top of the α1–α2
interface. The 1 : 1 stoichiometry of this asymmetric binding precludes the
linkage of one IgE to two receptor molecules and ensures that triggering
due to α–α aggregation only occurs through multivalent binding to surface
IgE (see Figure 15.2). (Photograph kindly provided by Ted Jardetzky and
reproduced by permission of the Nature Publishing Group.)
Figure 15.2. Clustering of IgE receptors
Cross-linking of FceRI by binding of multivalent allergen to IgE sensitizing
the mast cell leads to degranulation. Note that the two antibodies are against
different epitopes on the same allergen, and therefore will need to be
represented on the mast cell surface at a reasonably high frequency in order
for efficient cross-linking to occur.
Clinical responses to extrinsic allergens
It has been claimed that in westernized countries up to 30% of adults and
45% of children may suffer to a greater or lesser degree with allergies
involving localized IgE-mediated reactions to allergens such as grass
pollens, animal danders, the feces from mites in house dust (Figure 15.5)
and so on. Even if these are overestimates, it is clear that allergies affect a
large number of people and that they are on the increase. A large number of
allergens have now been cloned and expressed (Table 15.2), several of
which turn out to be enzymes. For example, Der p 1 is a cysteine protease
that increases the permeability of the bronchial mucosa, thereby facilitating
its own passage along with other allergens across the epithelium and
allowing access to and sensitization of cells of the immune system. The
CD23 low affinity receptor for IgE (FcsRII) on B-cells downregulates IgE
synthesis upon antigen-mediated cross-1 inking of the bound IgE. However,
Der p 1 proteolytically cleaves CD23 and thereby reduces its negative
impact on IgE synthesis. Furthermore, Der p 1 also cleaves CD25 (the IL-2
receptor α chain) on T-cells and thus limits the activation of Th1 cells,
biasing the immune response to Th2-dependent IgE production. Short cuts
to allergen purification can be achieved by screening cDNA expression
libraries with IgE. This was a godsend for the purification of the allergen
from the venom of the Australian jumper ant, Myrmecia pilosula; just think
of trying to accumulate ants by the kilogram to isolate the allergen using
conventional protein fractionation.
Figure 15.3. Mast cell triggering.
Simplified scheme of some of the signaling events through the high affinity
IgE receptor, FcεRI. Aggregation of the FcεRI a chains in lipid rafts
through cross-linking of bound IgE by multivalent antigen (allergen) leads
to ITAMs in the β and γ chains of the receptor interacting with the Lyn, Syk
and Fyn protein tyrosine kinases. Phosphorylation of Syk leads to its
activation and it in turn phosphorylates and activates the membrane adaptor
LAT1 and LAT2 (NTAL) proteins that recruit phospholipase Cγ1 (PLCγl)
and adaptor molecules concerned in the activation of GTPase/kinase
cascades. Activation of PLCγl generates diacylglycerol (DAG) that targets
protein kinase C, while inositol 1,4,5-triphosphate (IP ) elevates
3
cytoplasmic Ca 2+ by depleting the ER stores. The raised calcium
concentration activates transcriptional factors and causes granule
exocytosis. The Grb-2/Sos and Slp-76/Vav complexes associate with the
LAT1 adaptor, and Grb-2/Sos additionally with LAT2, and trigger the Ras
GTPase-induced serial kinase cascade leading to the activation of
transcription factors and rearrangements of the actin cytoskeleton. (Figure
essentially designed by Helen Turner, based on the article by Turner H. &
Kinet J.-P (1999) Nature (Supplement on Allergy and Asthma) 402, B24.)
The local anaphylactic reaction to injection of antigen into the skin of
atopic patients is manifest as a weal and flare (Figure 15.6), which is
maximal at 30 minutes or so and resolves within about an hour; it may be
succeeded by a late phase response involving eosinophil infiltrates that peak
at around 5 hours. Contact of the allergen with celhbound IgE in the
bronchial tree, the nasal mucosa and the conjunctival tissues releases
mediators of anaphylaxis and produces the symptoms of asthma or allergic
rhinitis and conjunctivitis (hay fever) as the case may be. A proportion of
the patients who experience late phase responses after bronchial challenge
with allergen eventually develop chronic asthma. Three hundred million
individuals worldwide suffer from asthma and it costs over US$6 billion a
year to treat in the USA alone. Indeed, according to the World Health
Organization, the worldwide economic costs associated with asthma are
estimated to exceed those of tuberculosis and HIV/AIDS combined.
Asthma can be associated with agents encountered in the workplace, and is
then described as occupational asthma. Allergens here include toluene
diisocyanate in spray paints, colophony fumes from solders used in the
electronics industry and danders (particles of old skin on animal hair)
encountered by animal handlers. Although the majority of asthma patients
have extrinsic asthma associated with atopy (from the Greek atopos,
meaning “out of place”-, i.e. the genetic predisposition to synthesize
inappropriate levels of IgE specific for external allergens, some patients are
nonatopic and therefore are said to have intrinsic or idiopathic asthma.
Figure 15.4. The allergy march.
In many children there is a temporal progression in the development of
allergies. (Modified with kind permission from a figure produced by Ulrich
Wahn for the World Allergy Organization (http://www.worldallergy.org)).
Figure 15.5. House dust mite—a major cause of allergic disease. The
electron micrograph shows the rather nasty looking mite graced by the
name Dermatophagoides pteronyssinus and fecal pellets on the bottom left.
A typical double bed can contain up to 200 million mites, each mite
producing approximately 20 fecal pellets/day and each pellet containing 0.2
ng of proteolytically active Der p 1 allergen. The biconcave pollen grains
(top left) shown for comparison indicate the size of particle that can become
airborne and reach the lungs. The mite itself is much too large for that.
(Reproduced by courtesy of E. Tovey.)
Figure 15.6. Atopic allergy.
Skin prick tests with grass pollen allergen in a patient with typical summer
hay fever. Skin tests were performed 5 hours (left) and 20 minutes (right)
before the photograph was taken. The tests on the right show a typical
titration of a type I immediate weal and flare reaction. The late phase skin
reaction (left) can be clearly seen at 5 hours, especially where a large
immediate response has preceded it. Figures for allergen dilution are given.
Bronchial biopsy and lavage of asthmatic patients reveal an unequivocal
involvement of mast cells and eosinophils as the major mediator-secreting
effector cells, while T-cells provide the microenvironment required to
sustain the chronic inflammatory response, which is an essential feature of
the histopa-thology (Figure 15.7). The resulting variable airflow obstruction
and bronchial hyper-sesponsiveness are the cardinal clinical and
physiological features of the disease.
The atopic trait can also manifest itself as an atopic dermatitis (eczema)
(Figure 15.8), with house dust mite, domestic cats and German cockroaches
often proving to be the environmental offenders. Recalling the
inflammation in asthma, skin patch tests with Der p 1 in these eczema
patients produce an infiltrate of eosinophils, T-cells, mast cells and
basophils. The number of individuals affected is comparable to the number
affected by asthma. The beneficial effect of the calcineurin inhibitors
cyclosporine and, more recently, topical tacrolimus in patients with eczema
highlights the important role of T-cells in the pathogenesis of this disease.
Awareness of IgE sensitization to food allergens in the gut has increased
dramatically. Contact with allergens such as those present in cows’ milk,
eggs, nuts and shellfish before mucosal protective mechanisms, especially
IgA, are reasonably established leads to an increase in the incidence of
atopy in the newborn (Figure 15.4). Although, overall, children who are
breastfed have a lower incidence of allergies, sensitization to dietary
allergens can also occur in early infancy through breastfeeding, with
antigen passing into the mother’s milk. One could argue that breastfeeding
mothers should limit their intake of common allergens. Allergy to peanuts is
seen in approximately 1% of children and, as with other allergens, reactions
are sometimes life threatening or even occasionally fatal. Food additives
such as sulfiting agents can also cause adverse reactions. Contact of the
food with specific IgE on mast cells in the gastrointestinal tract may
produce local reactions resulting in abdominal pain, cramps, diarrhea and
vomiting, or may allow the allergen to enter the body by causing a change
in gut permeability through mediator release; the allergen may complex
with antibodies and cause distal lesions by depositing in the joints, for
example, or it may diffuse freely to other sensitized sites, such as the skin
(Figure 15.8) or lungs, where it will cause a further local anaphylactic
reaction. Thus eating strawberries may produce urticarial reactions (hives,
raised areas of itchy skin) and egg may precipitate an asthmatic attack in
appropriately sensitized individuals. The role of the sensitized gut in acting
as a “gate” to allow entry of allergens is strongly suggested by experiments
in which oral sodium cromoglycate, a mast cell stabilizer, prevented
subsequent asthma after ingestion of the provoking food (Figure 15.9).
Table 15.2. Some examples of allergens.
Anaphylactic drug allergy is manifest in the dramatic responses to drugs
such as penicillin) which haptenate body proteins by covalent coupling to
induce IgE synthesis. In the case of penicillin, the P4actam ring links to the
e-amino of lysine to form the penicilloyl determinant. The fine specificity
of the IgE antibodies permits discrimination between closely similar drugs,
such that some patients may be allergic to amoxi-cillin but tolerate
benzylpenicillin, which differs by only very minor modifications of the side
chains.
Pathological mechanisms in asthma
We should now look in more depth at those events that generate the
chronicity of asthma. Remember that there is an early phase bronchial
response to inhaled antigen essentially involving mast cell mediators, and
an inflammatory late phase dominated by eosinophils. Both phases are
IgE-dependent as shown by their marked attenuation in asthmatics treated
with the humanized monoclonal anti-I gE antibody Omalizumab, which
reduces IgE to almost undetectable levels. Activated mast cells produce IL-
11, which contributes towards the development of the asthma-associated
structural changes referred to as airway remodeling- thickening of the
airway walls, and increases in the adventitia (the outermost connective
tissue), submucosal tissue and smooth muscle. The mast cells also
contribute to eosinophil recruitment by secretion of tryptase, which can
activate coagulation Factor II receptor-like 1 (F2RL1, protease-activated
receptor-2 [PAR-2]) on the surface of endothelial and epithelial cells,
fibroblasts and
Figure 15.7. Pathological changes in asthma.
Diagram of cross-section of an airway in severe asthma.
Figure 15.8. An atopic eczema reaction on the back of a knee of a child
allergic to rice and eggs.
(Kindly provided by J. Brostoff.)
Figure 15.9. The role of gut sensitivity in the development of asthma to
food allergens.
smooth muscle. Activation of the receptor leads to TNF, IL-1 and IL-4
production, promoting the expression of the vascular endothelial adhesion
molecules VCAM-1, ICAM-1 and β-selectin, which recruit eosinophils and
basophils. An important trigger of the late phase reaction is the activation
of alveolar macrophages through the interaction of allergen with IgE
bound to the low affinity FceRII leading to a significant increase in the
production of TNF and IL-1β. These cytokines stimulate the release of the
powerful eosinophil chemoattractants CCL5 (RANTES), CCL11
(eotaxin), and CCL12 (MCP5) ( cf. p. 232) from bronchial epithelial cells
and fibroblasts. Note also that CCL5 and CCL11 can contribute directly to
local inflammation by IgE-independent degranulation of basophils.
A patient challenged by feeding with egg developed asthma within hours,
as shown here by the depressed lung function test of measuring peak air
flow; the symptoms at the end-organ stage were counteracted by the ß-
adrenoreceptor agonist, isoprenaline. However, oral sodium cromoglycate
(SCG), which prevents antigen-specific mast cell triggering, also prevented
the onset of asthma after oral challenge with egg. Note that SCG taken
orally has no effect on the response of an asthmatic to inhaled allergen.
(From Brostoff J. (1986), In Brostoff J. & Challacombe S.J. (eds) Food
Allergy, p. 441. Bailliere Tindall, London, reproduced with permission.)
A new player now enters the field: primed T-cells traffic into the inflamed
site under the influence of CCL11. The GATA-3 transcription factor, c-maf
and the presence of pros-taglandin E-all promote Th2 development- and
responses are heavily skewed towards this particular T-cell subset in
asthma (Figure 15.10). Encounter with allergen-derived pep-tides on
antigen-presenting cells will promote the synthesis of IL-4, -5 and -13. IL-4
stimulates further CCL11 release, while IL-5 upregulates chemokine
receptors on eosinophils, maintains their survival through an inhibitory
effect on natural apoptosis and is involved in their longer term recruitment
from bone marrow. Th17 cells are also present and promote both neutrophil
and macrophage inflammatory responses in the lung.
Figure 15.10. Th2 dominance in atopic allergy.
Shown by cytokine profiles of antigen-specific CD4 T-cell clones from (a)
patients with type I atopic allergy and (b) subjects with type IV contact
sensitivity, compared with normal controls. Each point represents the value
for an individual clone. Archetypal Th1 clones have high IFNγ and IL-2
and low IL-4 and IL-5; Th2 clones show the converse. The high level of IL-
4 drives the switch to IgE production by B-cells and further promotes the
Th2 bias. (Data from Kapsenberg M.L., Wierenga E.A., Bos J.D. & Jansen
H.M. (1991) Immunology Today 12, 392.)
Things now look bad for the bronchial tissues and a multitude of factors
contribute to allergen-induced airway dysfunction: (i) a virtual soup of
bronchoconstrictors, the leukotrienes being especially important, bathe the
smooth muscle cells; (ii) edema of the airway wall; (iii) altered neural
regulation of airway tone through binding of eosinophil major basic protein
(MBP) to M2 autoreceptors on the nerve endings with increased release of
acetylcholine; (iv) airway epithelial cell desquamation due to the toxic
action of MBP, there being a strong correlation between the number of
desquamated cells in bronchoalveolar lavage fluid and the concentration of
MBP; (v) mucus hypersecretion due to IL-13 and, to a lesser extent, IL-4,
leukotrienes and platelet activating factor acting on sub-mucosal glands and
their controlling neural elements; and finally (vi) a repair-type response
involving the production of fibroblast growth factor, TGFPβ and platelet-
derived growth factor, the laying down of collagen, scar and fibrous tissue
and hypertrophy of smooth muscle, leading to an exaggerated narrowing of
the airways in response to a variety of environmental stimuli (Figure 15.7).
The wide range of cytokines and mediators produced by lung epithelial and
endothelial cells, fibrob-lasts and smooth muscle cells may account for the
persistence of airway inflammation and the permanent structural changes in
chronic disease sufferers, even in the absence or apparent absence of
ongoing exposure to inhalant allergens to which subjects are sensitized, a
state where conventional immuno-therapy might not be expected to be
beneficial.
Unlike atopic asthmatics, intrinsic asthmatics have negative skin tests to
common aeroallergens, no clinical or family history of allergy, normal
levels of serum IgE and no detectable specific IgE antibodies to common
allergens. Nonetheless, they resemble the atopics in important respects:
bronchial biopsies show enhanced expression of IL-4, IL-13, CCL5 and
CCL11, and of the mRNA for the e heavy chain, suggestive of local IgE
synthesis. Is there a role for virus-specific IgE or for IgE autoantibodies to
the FcεRI?
The inflammatory infiltrate in atopic dermatitis resembles that in asthma
and includes mast cells, basophils, eosinophils and T-cells. Epidermal
dendritic cells express FceRI, and incoming allergens are taken up as
allergen—IgE complexes and passed to the MHC class II processing
pathway for presentation to Th2 cells. CC chemokines produced by
keratinocytes and fibroblasts preferentially attract eosinophils and the skin-
homing CLA+ memory Th2-cells. The latter comprise 80–90% of the
T)Cells in the infiltrate and account for the specific response to the
offending allergen.
Etiological factors in the development of atopic
allergy
Thiere is a strong familial predisposition to the development of atopic
allergy (Figure 15.11) suggestive of genetic factors. Indeed, it is clear that
the development of atopic allergy depends upon complex multiple genetic
interactions with various environmental factors. Age, sex, infection history,
nutritional status and allergen exposure all play a role. One obvious factor is
the overall ability to synthesize the IgE isotype—the higher the level of IgE
in the blood, the greater the likelihood of becoming atopic (Figure 15.11).
Genetic studies have provided evidence that many different genes
contribute to susceptibility to develop asthma (Figure 15.12) although no
one single gene is a particularly strong predisposing factor on its own. One
interesting association, however, is with polymorphisms in a number of
pattern recognition receptors (PRR). What relevance might this have for
atopic disease? Well, the PRR-mediated recognition of pathogens by
dendritic cells is important in developing the correct balance between Th1
and Th2 responses. Current thinking goes along the following lines. At the
time of birth the neonatal immune system is skewed towards Th2-ype
responses, but in the face of a hostile microbial environment there is a shift
towards Th1 responses. This shift extends to inhaled allergens, and is
sometimes referred to as immune deviation. However, in the absence of
repeated infections with common pathogens (due to a “cleaner”
environment and widespread early use of antibiotics) the immune system
maintains a Th2 phenotype, which will favor the secretion of IL-4
(promoting IgE production) and IL-5 (promoting eosinophilia). This idea
forms the basis of the hygiene hypothesis put forward to explain the rise in
allergies seen in highly developed countries, and even more tellingly in
countries that become highly developed, such as the former East Germany
where levels of atopic allergy started to catch up with those in West
Germany following re-unification. The overall picture relating to economic
development is, however, complex—kt us not forget that a finger has been
pointed at environmental pollutants such as diesel exhaust particles as
cofactors for asthma attacks.
Figure 15.11. Risk factors in allergy
(a) Family history; (b) IgE levels—the higher the serum IgE concentration,
the greater the chance of developing atopy.
Recently there has also been a great deal of interest in trying to
understand the role of the barrier function of the epithelium in allergic
responses. Compromise of the normally tight junctions between the
epithelial cells, perhaps caused by chemical or physical pollutants or by
infection, will clearly lead to increased access of both pathogens and
allergens. Yet another piece of the jigsaw relates to the role of regulatory T-
cells (Tregs) in atopic disease. Evidence is accumulating that indicates a
deficit of these cells in patients with allergy, with both naturally-occuring
+
+
+
CD4 CD25 Foxp3 Tregs and inducible Tregs being implicated. The
Tregs may themselves be influenced by interactions with distinct dendritic
cell subsets. And where do Th17 cells fit into all of this? Watch this space,
as they say.
Clinical tests for allergy
Sensitivity is normally assessed by the response to intradermal challenge
with antigen. The release of histamine and other mediators rapidly produces
a weal and erythema (see Figure 15.6), maximal within 30 minutes and
then subsiding. These immediate weal and flare reactions may be followed
by a late phase reaction (see Figure 15.6), which sometimes lasts for 24
hours, redolent of those seen following challenge of the bronchi and nasal
mucosa of allergic subjects and similarly characterized by dense infiltration
with eosinophils and T-cells.
The correlation between skin prick test responses and the
radioallergosorbent test (RAST, see p. 167) for allergen-specific serum
IgE is fairly good. In some instances, intranasal challenge with allergen
may provoke a response even when both of these tests are negative,
probably as a result of local synthesis of IgE antibodies.
Figure 15.12. Gene products that influence susceptibility to asthma.
Multiple genes have been implicated that act at various stages in the type I
hypersensitivity response. ADAM33, disintegrin and metalloprotein
domain-containing protein 33; DPP10, dipeptidyl peptidase 10; NPSR1,
neuropeptide S receptor 1; PCDH1, protocadherin-1; PTGDR,
prostaglandin D receptor; TIM1, T-cell, immunoglobulin and mucin
2
domain-containing protein 1. (Modified from Cookson W. & Moffatt M.
(2004) New England Journal of Medicine 351 , 1794–1796, with
permission from the publishers.)
The presence of proteins secreted from mast cells or eosinophils in the
serum or urine could provide important surrogate markers of disease and
might predict exacerbations.
Therapy
If one considers the sequence of reactions from initial exposure to allergen
right through to the production of atopic disease, it can be seen that several
points in the chain provide legitimate targets for therapy (Figure 15.13).
Allergen avoidance. Avoidance of contact with potential allergens is often
impractical, although, to give one example, feeding cows’ milk to infants at
too early an age is discouraged. After sensitization, avoidance where
possible is obviously worthwhile, but the reluctance of some parents to
dispose of the family cat to stop little Algernons wheezing is sometimes
quite surprising.
Modulation of the immunological response. Desensitization by repeated
subcutaneous injection of small amounts of allergen can lead to
worthwhile improvement in individuals subject to insect venom
anaphylaxis or hay fever. Sublingual allergen immunotherapy (SLIT) is
less time consuming for the patient and carries less risk of severe systemic
reactions than subcutaneous administration; but this has to be balanced
against the fact that it is sometimes not quite as effective as injection
immuno-therapy. The purpose of allergen hyposensitization therapy was
originally to boost the synthesis of IgG “blocking” antibodies, whose
function was to divert the allergen from contact with tissue-bound IgE.
While this may well be a contributory factor, downregulation of IgE
synthesis by engagement of the FcγRIIB receptor (cf. p. 264) on B-cells by
allergen-specific IgG linked to allergen molecules bound to surface IgE
receptors also seems likely (cf. p. 264; see Chapter 10 on IgG regulation of
Ab production). Additionally, T-lymphocyte cooperation is important for
IgE synthesis and eosinophil-mediated pathogenesis, and therefore the
beneficial effects of antigen injection may also be mediated through
induction of anergic or regulatory T-cells and a switch from Th2 to Th1
cytokine production. Injection of heat-killed Mycobacterium vaccae
induces IL-10 and TGFP secretion by regulatory T-cells with a resultant
decrease in Th2 activity. Inhibition of the Th2-associated transcription
factor GATA-3 using PPAR (peroxisome proliferator-activated receptor)
agonists, or stimulating Th1-associated T-bet expression with CpG motifs,
may provide future therapeutic options for promoting Th1 rather than Th2
responses. The administration of tolerizing or antagonist peptide epitopes
represents another possible therapeutic modality. Fortunately, most patients
respond to a remarkably limited number ofT-cell epitopes on any given
allergen, and so it may not be necessary to tailor the therapeutic peptide to
each individual. Clinical trials of immunotherapy with Fel d 1-derived
peptides from cat allergen have resulted in a decrease of both early and late-
phase reactions. A case can be made for future prophylactic
hyposensitization of children with two asthmatic parents given that such
youngsters have an approximately 50% probability of developing the
disease.
Figure 15.13. Atopic allergies and their treatment: sites of local
responses and possible therapies.
Events and treatments relating to local anaphylaxis are in green and to
chronic inflammation in red. MAb, monoclonal antibody.
Blocking the action of IgE. We have already mentioned the humanized
monoclonal Omalizumab directed against the FceRI-binding Ce3 domain
ofIgE (cf. p. 396), which provides an exciting new therapy for severe forms
of asthma. It reduces the circulating IgE levels almost to vanishing point by
direct neutralization, and as a secondary effect this decreases the IgE-
dependent expression of the FceRI receptor on mast cells. Thus there are far
fewer receptors on the mast cell to bind IgE, and virtually no IgE to be
bound anyway. It is not surprising therefore that this antibody successfully
completed phase II clinical trials and was subsequently approved by the
FDA for use in those adults and adolescents with moderate or severe
persistent atopic asthma whose symptoms are inadequately controlled with
inhaled corticosteroids.
Stabilization of the triggering cells. Much relief has been obtained with
agents such as inhalant isoprenaline and sodium cromoglycate (cromolyn
sodium), which render mast cells resistant to triggering. Sodium
cromoglycate blocks chloride channel activity and maintains cells in a
normal resting physiological state, which probably accounts for its
inhibitory effects on a wide range of cellular functions, such as mast cell
degranulation, eosinophil and neutrophil chemotaxis and mediator release,
and reflex bronchoconstriction. Some or all of these effects are responsible
for its anti-asthmatic actions.
The triggering of macrophages through allergen interaction with surface-
bound IgE is clearly a major initiating factor for late reactions, as discussed
above, and resistance to this stimulus can be very effectively achieved with
corticosteroids. Unquestionably, inhaled corticosteroids have
revolutionized the treatment of asthma. Their principal action is to suppress
the transcription of multiple inflammatory genes, including in the present
context those encoding several cytokines.
Mediator antagonism. Histamine H 1-receptor antagonists have for
long proved helpful in the symptomatic treatment of atopic disease. Newer
drugs of this class such as loratadine and fexofenadine are effective in
rhinitis and in reducing the itch in atopic dermatitis, although they have
little benefit in asthma. Cetirizine additionally has useful effects on
eosinophil recruitment in the late phase reaction. Short-acting selective β -
2
agonists such as Ventolin, the active ingredient of which is albuterol
(salbutamol), are inhaled to alleviate mild-to-moderate symptoms of
asthma. Such β-adrenergic receptor agonist drugs increase cAMP levels
leading to relaxation of bronchial smooth muscle and inhibition of mast cell
degranulation. An important advance has been the introduction of long-
acting β -agonists such as salmeterol and formoterol, which protect
2
against bronchoconstriction for over 12 hours. Potent leukotriene receptor
antagonists such as pranlukast also block constrictor challenges and show
striking efficacy in certain patients, particularly aspirin-sensitive asthmatics.
Figure 15.14. Antibody-dependent cytotoxic hypersensitivity (type II).
Antibodies directed against cell surface antigens cause cell death not only
by complement-dependent lysis using the C5b-C9 membrane attack
complex (MAC) but also by Fcγ and C3b adherence reactions leading to
phagocytosis, or through nonphagocytic extracellular killing by antibody-
dependent cellular cytotoxicity (ADCC). Human monocytes and IFNγ-
activated neutrophils kill Ab-coated tumor cells using their FcγRI receptors;
NK cells kill targets through FcγRIII receptors.
Theophylline was introduced for the treatment of asthma over 60 years
ago and, as a phosphodiesterase (PDE) inhibitor, it increases intracellular
cAMP, thereby causing bronchodilatation. Generally good news for the
patient, although concern over some of its side effects mean that it is often
only used when other treatment options prove ineffective.
Attacking chronic inflammation. Certain drugs impede atopic disease at
more than one stage. Cetirizine is a case in point with its dual effects on the
histamine receptor and on eosinophil recruitment. Corticosteroids seem to
do almost everything; apart from their role in stabilizing macrophages, they
solidly inhibit the activation and proliferation of Th2 cells, which are the
dominant underlying driving force in chronic asthma, and may call a halt to
the development of irreversible narrowing of the airways. So it is that
inhaled steroids (e.g. budesonide, mometasone furoate, fluticasone
propionate) with high anti-inflammatory potency but minimal side-effects
due to hepatic metabolism, provide first-line therapy for most chronic
asthmatics, with supplementation by long-acting P -agonists.
2
Antibody-dependent cytotoxic
hypersensitivity (type II)
Where an antigen is present on the surface of a cell, combination with
antibody will encourage the demise of that cell by promoting contact with
phagocytes by opsonic adherence to Fcγ receptors and, often, to C3b
receptors following activation of complement by the classical pathway. Cell
death may also occur through activation of the full complement system up
to C8 and C9 producing direct membrane damage (Figure 15.14),
although this will have to overcome the protective effect of cell surface
complement regulatory proteins.
A quite distinct cytotoxic mechanism, antibody-dependent cellular
cytotoxicity (ADCC), occurs when target cells coated with antibody are
killed through an extracellular nonphago-cytic process involving leukocytes
that bind to the target by their specific Fc receptors, e.g. FcγR in the case of
IgG (Figure 15.14). ADCC can be mediated by a number of different types
of leukocyte including NK cells (see p. 26), monocytes, neu-trophils and
eosinophils. Although readily observed as a phenomenon in vitro, e.g.
schistosomules coated with either IgG or IgE can be killed by eosinophils
(cf. Figure 12.25), whether ADCC plays a role in vivo remains a tricky
question. Functionally this extracellular cytotoxic mechanism would be
expected to be of significance where the target is too large for ingestion by
phagocytosis, e.g. large parasites and solid tumors. It could also act as a
back-up system for T-cell mediated killing.
Type II reactions between members of the same
species (alloimmune)
Transfusion reactions
Of the many different polymorphic constituents of the human red cell
membrane, ABO blood groups form the dominant system. The antigenic
groups A and B are derived from H substance (Figure 15.15) by the action
of glycosyltransferases encoded by A or B genes, respectively. Individuals
with both genes (group AB) have the two antigens on their red cells, while
those lacking these genes (group O) synthesize H substance only.
Antibodies to A or B occur spontaneously when the antigen is absent from
the red cell surface; thus a person of blood group A will possess anti-B and
so on. These isohemag-glutinins are usually IgM and probably belong to
the class of “natural antibodies”; they would be boosted through contact
with antigens of the gut flora that are structurally similar to the blood group
carbohydrates, so that the antibodies formed cross-react with the
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